Changes in catalytic activity and association state of pyruvate carboxylase which are dependent on enzyme concentration

The specific activity of chicken liver pyruvate carboxylase has been shown to decrease with decreasing enzyme concentration, even at 100 microM, which is close to the estimated physiological concentration. The kinetics of the loss of enzyme specific activity following dilution were biphasic. Incubation of dilution-inactivated enzyme with ATP, acetyl CoA, Mg2+ + ATP or, to a lesser degree, with Mg2+ alone resulted in a high degree of reactivation, while no reactivation occurred in the presence of pyruvate. The association state of the enzyme before, during, and after dilution inactivation has been assessed by gel filtration chromatography. These studies indicate that on dilution, there is dissociation of the catalytically active tetrameric enzyme species into inactive dimers. Reactivation of the enzyme resulted in reassociation of enzymic dimers into tetramers. The enzyme was shown to form high molecular weight aggregates at high enzyme concentrations.     

Dissociation of the octameric bifunctional enzyme formiminotransferase-cyclodeaminase in urea. Isolation of two monofunctional dimers

Partial denaturation of the circular octameric bifunctional enzyme formiminotransferase-cyclodeaminase in increasing urea concentrations leads to sequential dissociation via dimers to inactive monomers. In potassium phosphate buffer, dissociation to dimers in 3 M urea coincides with loss of both activities and a major decrease in intensity of intrinsic tryptophan fluorescence. In the presence of folic acid, these dimers retain the deaminase activity, but with folylpolyglutamates, both activities are protected and the native octameric structure is retained. The protection profiles with polyglutamates are cooperative with a Hill coefficient greater than 2, suggesting that binding of more than one folylpolyglutamate per octamer is required to stabilize the native structure. In triethanolamine hydrochloride buffer, transferase-active dimers that retain the intrinsic tryptophan fluorescence can be obtained in 1 M urea and stabilized at higher urea concentration by the addition of glutamate. Deaminase-active dimers are obtained by the protection of folate in 3 M urea. Proteolysis of the two kinds of dimers by chymotrypsin leads to very different fragmentation patterns, indicating that they are structurally different. We propose that the two dimers retain different subunit-subunit interfaces, one of which is required for each activity. This suggests that the native octameric structure is required for expression of both activities and therefore for "channeling" of intermediates.     

Folding and stability of different oligomeric states of thiamin diphosphate dependent homomeric pyruvate decarboxylase

The folding and stability of recombinant homomeric (alpha-only) pyruvate decarboxylase from yeast was investigated. Different oligomeric states (tetramers, dimers and monomers) of the enzyme occur under defined conditions. The enzymatic activity is used as a sensitive probe for structural differences between the active and inactive form (mis-assembled forms, aggregates) of the folded protein. Unfolding kinetics starting from the native protein comprise both the dissociation of the oligomers into monomers and their subsequent denaturation, which could be monitored by stopped-flow kinetics. In the course of unfolding, the tetramers do not directly dissociate into monomers, but via a stable dimeric state. Starting from the unfolded state, a reactivation of homomeric pyruvate decarboxylase requires both refolding to monomers and their correct association to enzymatically active dimers or tetramers. The reactivation yield under the in vitro conditions used follows an optimum behavior.     

Active oligomeric states of pyruvate decarboxylase and their functional characterization.

Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimers and tetramers under physiological conditions, a K(d) value of 8.1 microM was determined by analytical ultracentrifugation. Dimers and monomers of the enzyme could be populated by equilibrium denaturation using urea as denaturant at defined concentrations and monitored by a combination of optical (fluorescence and circular dichroism) and hydrodynamic methods (analytical ultracentrifugation). Dimers occur after treatment with 0.5 M urea, monomers with 2.0 M urea independent of the protein concentration. The structured monomers are catalytically inactive. At even higher denaturant concentrations (6 M urea) the monomers unfold. The contact sites of two monomers in forming a dimer as the smallest enzymatically active unit are mainly determined by aromatic amino acids. Their interactions have been quantified both by structure-theoretical calculations on the basis of the X-ray crystallography structure, and experimentally by binding of the fluorescent dye bis-ANS. The contact sites of two dimers in tetramer formation, however, are mainly determined by electrostatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruvate. There was no difference in the steady-state activity (specific activity) between dimers and tetramers. The activation kinetics of the two oligomeric states, however, revealed differences in the dissociation constant of the regulatory substrate (K(a)) by one order of magnitude. The tetramer formation is related to structural consequences of the interaction transfer in the activation process causing an improved substrate utilization.     

Equilibrium studies on the refolding and reactivation of rabbit-muscle aldolase after acid dissociation.

Dissociation, denaturation, and deactivation of aldolase from rabbit muscle in the acid pH range have been investigated using sedimentation analysis, fluorescence, circular dichroism, and activity tests. Under comparable experimental conditions the pH-dependent profiles of deactivation and denaturation parallel the dissociation of the enzyme. In the range of dissociation at pH4-5tetramers and monomers are in equilibrium. Intrinsic chromophores and far-ultraviolet circular dichroism suggest the transition to be a complex multistep process. At pH approximately 2.3 the enzyme is split into its fully inactive monomers which still contain some residual secondary structure. After reassociation under optimum conditions (0.2 M phosphate buffer pH 7.6, 1 mM EDTA, 0.1 mM dithiothreitol, 0 degrees C, enzyme concentration 0.4-59 mug/ml) up to 95% enzymic activity is recovered which belongs to a renatured tetrameric species indistinguishable from the native enzyme by all available biochemical and physicochemical criteria.     

Dissociation and in vitro reconstitution of bovine liver uridine diphosphoglucose dehydrogenase. The paired subunit nature of the enzyme

Uridine diphosphoglucose dehydrogenase (EC 1.1.1.22: UDPglucose dehydrogenase) at pH 5.5-7.8 is a stable homohexamer of 305 +/- 7 kDa that does not undergo concentration-dependent dissociation at enzyme concentrations greater than 5 micrograms/mL. Chemical cross-linking of the native enzyme at varying glutaraldehyde concentrations yields dimers, tetramers, and hexamers; at greater than 2% (w/v) glutaraldehyde, plateau values of 21% monomers, 16% dimers, 5% tetramers, and 58% hexamers are obtained. Dissociation at acid pH (pH 2.3) or in 4-6 M guanidine hydrochloride leads to inactive monomers (Mr 52,000). Denaturation at increasing guanidine hydrochloride concentration reveals separable unfolding steps suggesting the typical domain structure of dehydrogenases holds for the present enzyme. At greater than 4 M guanidine hydrochloride complete randomization of the polypeptide chains is observed after 10-min denaturation. Reconstitution of the native hexamer after dissociation/denaturation has been monitored by reactivation and glutaraldehyde fixation. The kinetics may be described in terms of a sequential uni-bimolecular model, governed by rate-determining folding and association steps at the monomer level. Trimeric intermediates do not appear in significant amounts. Reactivation is found to parallel hexamer formation. Structural changes during reconstitution (monitored by circular dichroism) are characterized by complex kinetics, indicating the rapid formation of "structured monomers" (with most of the native secondary structure) followed by slow "reshuffling" prior to subunit association. The final product of reconstitution is indistinguishable from the initial native enzyme.     

Active subunits of rabbit liver fructose diphosphatase

Fructose diphosphatase, bound to a matrix of Sepharose, retains most of the catalytic activity but becomes half desensitized to AMP. The dimers, obtained by acid dissociation of the enzyme bound to the matrix, possess half of the specific activity of the tetramers and are almost completely desensitized to AMP. The monomers are inactive.     

Structural organization of glutamate dehydrogenase. 2. Inactivation and dissociation of immobilized hexamer induced by urea

The urea-induced inactivation and dissociation of catalytically active hexamer of glutamate dehydrogenase (L-glutamate-NAD(P)-oxidoreductase, EC 1.4.1.3) from bovine liver were studied using radioactive phosphopyridoxyl derivative of the enzyme immobilized on cyanogen bromide-activated Sepharose CL-4B. It is shown that at neutral pH (7.0-7.8) urea causes dissociation of glutamate dehydrogenase to directly yield catalytically inactive immobilized monomers rather than hexamer's stable fragments at the same time. At pH 8.9 or 5.6 the urea-induced is accompanied by the formation of conformationally stable immobilized dimers or trimers, respectively. The trimers are catalytically active, whereas the dimers did not exhibit any enzymatic activity. The data obtained led to suggestion that the hexamer consists of three either equivalent dimers (3 alpha 2) or of two equivalent trimers (2 alpha 3).     

Studies on dilution inactivation of sheep liver pyruvate carboxylase

When sheep liver pyruvate carboxylase was diluted below 4 EU/ml, it underwent inactivation involving two kinetically distinct processes, i.e., a rapid initial burst followed by a slower second phase. The catalytic activity of the diluted enzyme eventually approached zero, suggesting the occurrence of an irreversible process. Analysis of the quaternary structure of the enzyme by gel filtration chromatography and electron microscopy showed that most of the enzyme molecules occur as tetramers at high enzyme concentrations. However, dilution of the enzyme below 4 EU/ml led to the appearance of dimers and monomers which were essentially inactive under the conditions of the assay system used. The presence of acetyl-CoA during dilution prevented inactivation from occurring and preserved the tetrameric structure. When added after dilution, acetyl-CoA prevented further inactivation from occurring but did not reactivate the enzyme. However, acetyl-CoA did cause a relatively rapid reassociation of the inactive monomers and dimers to form inactive tetramers.     

Subunit interactions and immobilised dimers of human liver arginase.

Incubation of soluble human liver arginase (L-arginine amidinohydrolase, EC 3.5.3.1) with p-hydroxymercuribenzoate resulted in the dissociation of the enzyme into active dimers. Addition of 2-mercaptoethanol resulted in the regeneration of the tetrameric enzyme. When arginase, bound covalently to nylon, was incubated with p-hydroxymercuribenzoate, matrix-bound dimers were obtained. Incubation of these species with 2-mercaptoethanol resulted in stable, unmodified dimers. Based on this dissociation of arginase, a model with D2-symmetry is suggested for this enzyme. The specific activity, the Km value for arginine, pH optimum and the inhibition constants for ornithine and lysine were determined for monomeric, dimeric and tetrameric forms. It is concluded that the behaviour of the active sites of the monomers is not substantially altered by the interaction of these species in the oligomeric molecule.     

Nativelike intermediate on the unfolding pathway of pig kidney fructose-1,6-bisphosphatase

The unfolding and dissociation of the tetrameric enzyme fructose-1,6-bisphosphatase from pig kidney by guanidine hydrochloride have been investigated at equilibrium by monitoring enzyme activity, ANS binding, intrinsic (tyrosine) protein fluorescence, exposure of thiol groups, fluorescence of extrinsic probes (AEDANS, MIANS), and size-exclusion chromatography. The unfolding is a multistate process involving as the first intermediate a catalytically inactive tetramer. The evidence that indicates the existence of this intermediate is as follows: (1) the loss of enzymatic activity and the concomitant increase of ANS binding, at low concentrations of Gdn.HCl (midpoint at 0.75 M), are both protein concentration independent, and (2) the enzyme remains in a tetrameric state at 0.9 M Gdn.HCl as shown by size-exclusion chromatography. At slightly higher Gdn.HCl concentrations the inactive tetramer dissociates to a compact dimer which is prone to aggregate. Further evidence for dissociation of tetramers to dimers and of dimers to monomers comes from the concentration dependence of AEDANS-labeled enzyme anisotropy data. Above 2.3 M Gdn.HCl the change of AEDANS anisotropy is concentration independent, indicative of monomer unfolding, which also is detected by a red shift of MIANS-labeled enzyme emission. At Gdn.HCl concentrations higher than 3.0 M, the protein elutes from the size-exclusion column as a single peak, with a retention volume smaller than that of the native protein, corresponding to the completely unfolded monomer. In the presence of its cofactor Mg(2+), the denaturated enzyme could be successfully reconstituted into the active enzyme with a yield of approximately 70-90%. Refolding kinetic data indicate that rapid refolding and reassociation of the monomers into a nativelike tetramer and reactivation of the tetramer are sequential events, the latter involving slow and small conformational rearrangements in the refolded enzyme.     

Pyridoxal phosphate inhibits dynamic subunit interchange among serine hydroxymethyltransferase tetramers

Cytoplasmic serine hydroxymethyltransferase (cSHMT) is a tetrameric, pyridoxal phosphate (PLP)-dependent enzyme that catalyzes the reversible interconversion of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. The enzyme has four active sites and is best described as a dimer of obligate dimers. Each monomeric subunit within the obligate dimer contributes catalytically important amino acid residues to both active sites. To investigate the interchange of subunits among cSHMT tetramers, a dominant-negative human cSHMT enzyme (DNcSHMT) was engineered by making three amino acid substitutions: K257Q, Y82A, and Y83F. Purified recombinant DNcSHMT protein was catalytically inactive and did not bind 5-formyltetrahydrofolate. Coexpression of the cSHMT and DNcSHMT proteins in bacteria resulted in the formation of heterotetramers with a cSHMT/DNcSHMT subunit ratio of 1. Characterization of the cSHMT/DNcSHMT heterotetramers indicates that DNcSHMT and cSHMT monomers randomly associate to form tetramers and that cSHMT/DNcSHMT obligate dimers are catalytically inactive. Incubation of recombinant cSHMT protein with recombinant DNcSHMT protein did not result in the formation of hetero-oligomers, indicating that cSHMT subunits do not exchange once the tetramer is assembled. However, removal of the active site PLP cofactor does permit exchange of obligate dimers among preformed cSHMT and DNcSHMT tetramers, and the formation of heterotetramers from cSHMT and DNcSHMT homodimers does not affect the activity of the cSHMT homodimers. The results of these studies demonstrate that PLP inhibits dimer exchange among cSHMT tetramers and suggests that cellular PLP concentrations may influence the stability of cSHMT protein in vivo.     

Studies on the oligomeric state of isolated cytochrome oxidase using cross-linking reagents

(1) Sucrose gradient centrifugation of cytochrome oxidase in the presence of Triton X-100 gave one slowly sedimenting green band. After cross-linking with dithiobis(succinimidylpropionate) (DSP), two green bands were observed, one sedimenting like the control and the other one more rapidly. Only the slowly sedimenting band was observed if the cross-linker was cleaved by dithiothreitol before centrifugation. (2) The rapidly sedimenting band in the Triton-containing sucrose gradient is probably the internally cross-linked dimer of cytochrome oxidase; the one sedimenting slowly is the monomeric enzyme. (3) Cross-linking with DSP after monomerization yields a small fraction of internally cross-linked dimers in addition to the internally cross-linked monomers. Under similar conditions, but using the shorter cross-linker disuccinimidyl tartarate (DST), no dimers are detected. (4) Both DSP and DST cross-link the dimeric enzyme so that it could no longer be monomerized by centrifugation in Triton, unless the cross-link is cleaved. (5) Polypeptide analysis using two-dimensional gel electrophoresis of cross-linked dimers and monomers suggest that subunit VIb is involved in intermonomeric cross-linking of dimeric enzyme by DSP.     

Studies on inactivation of lipoprotein lipase: role of the dimer to monomer dissociation

Sedimentation equilibrium analysis demonstrated that preparations of bovine lipoprotein lipase contain a complex mixture of dimers and higher oligomers of enzyme protein. Enzyme activity profiles from sedimentation equilibrium as well as from gel filtration indicated that activity is associated almost exclusively with the dimer fraction. To explore if the enzyme could be dissociated into active monomers, 0.75 M guanidinium chloride was used. Sedimentation velocity measurements demonstrated that this treatment led to dissociation of the lipase protein into monomers. Concomitant with dissociation, there was an irreversible loss of catalytic activity and a moderate change in secondary structure as detected by circular dichroism. The rate of inactivation increased with decreasing concentrations of active lipase, but addition of inactive lipase protein did not slow down the inactivation. This indicates that reversible interactions between active species precede the irreversible loss of activity. The implication is that dissociation initially leads to a monomer form which is in reversible equilibrium with the active dimer, but which decays rapidly into an inactive form, and is therefore not detected as a stable component in the system.     

Purification and characterization of indolepyruvate decarboxylase. A novel enzyme for indole-3-acetic acid biosynthesis in Enterobacter cloacae.

Indolepyruvate decarboxylase, a key enzyme for indole-3-acetic acid biosynthesis, was found in extracts of Enterobacter cloacae. The enzyme catalyzes the decarboxylation of indole-3-pyruvic acid to yield indole-3-acetaldehyde and carbon dioxide. The enzyme was purified to apparent homogeneity from Escherichia coli cells harboring the genetic locus for this enzyme obtained from E. cloacae. The results of gel filtration experiments showed that indolepyruvate decarboxylase is a tetramer with an M(r) of 240,000. In the absence of thiamine pyrophosphate and Mg2+, the active tetramers dissociate into inactive monomers and dimers. However, the addition of thiamine pyrophosphate and Mg2+ to the inactive monomers and dimers results in the formation of active tetramers. These results indicate that the thiamine pyrophosphate-Mg2+ complex functions in the formation of the tetramer, which is the enzymatically active holoenzyme. The enzyme exhibited decarboxylase activity with indole-3-pyruvic acid and pyruvic acid as substrates, but no decarboxylase activity was apparent with L-tryptophan, indole-3-lactic acid, beta-phenylpyruvic acid, oxalic acid, oxaloacetic acid, and acetoacetic acid. The Km values for indole-3-pyruvic acid and pyruvic acid were 15 microM and 2.5 mM, respectively. These results indicate that indole-3-acetic acid biosynthesis in E. cloacae is mediated by indolepyruvate decarboxylase, which has a high specificity and affinity for indole-3-pyruvic acid.     

Analytical ultracentrifugation studies on chloroplastic fructose bisphosphatase

Analytical ultracentrifugation studies performed on spinach chloroplast fructose bisphosphatase show that the tetrameric oxidized (inactive) or reduced (active) enzyme dissociates into inactive dimers and monomers at alkaline pH. The dissociation process is, at least, partially reversible if the enzyme is dimeric. Moreover, the oxidized inactive tetrameric enzyme is less prone to dissociation into dimers and monomers than the reduced active tetramer. The irreversibility of the dissociation process may be explained by a sulfhydryl-disulfide interchange. Together with the findings from previously published sulfhydryl group titration experiments (J. Pradel et al., Eur. J. Biochem., 113 (1981) 507), the above results suggest that the activation of the oxidized tetramer involves the reduction of two inter-protomeric disulfide bonds.     

The arrangement of ribosomes in ribosome tetramers from hypothermic chick embryos

1. Ribosomes and the tetramer arrangement peculiar to the tissues of chick embryos exposed to low temperatures were separated by sucrose-density-gradient centrifugation, and the effects of variation of the concentrations of Mg(2+), Ca(2+) and K(+) studied. 2. Lowering of the Mg(2+) concentration from standard buffer conditions caused a reversible dissociation of tetramers into monomers and of these into subunits. 3. Ca(2+) replaced Mg(2+) in causing the re-formation of tetramers and monomers from subunits after dissociation in low Mg(2+) concentrations. 4. Ca(2+) also caused an almost complete conversion of monomers into dimers in the presence of Mg(2+). 5. The effect of Ca(2+) on the formation of dimers was abolished by pretreatment of the ribosomes with ribonuclease, but the re-formation of tetramers was unaffected. 6. Increase of the K(+) concentration from that of the standard buffer caused dissociation of monomers and dimers into subunits. 7. Raised K(+) concentration also caused a stepwise alteration of the tetramer from a particle with a sedimentation coefficient of 197S, which constitutes the bulk of the tetramer at low K(+) concentrations, first to a 184S peak and finally to material with a sedimentation coefficient of about 155S. 8. The implications of these results on hypotheses of the arrangement of the individual monomers in the tetramer are discussed and a new model for the structure is proposed.     

Presence of SH-groups and histidine in the active site of Chlorella glutamine synthetase]

The presence of two cysteine residues per each six monomers comprising the oligomer of Chlorella glutamine synthetase (E.C.6.3.1.2) is demonstrated using homogenous enzyme preparation. p-Chloromercuribenzoate (p-CMB) is found to inhibit glutamine synthetase activity, the degree of inhibition depending on the inhibitor concentration. The following enzyme reactivation by dithiotreitol (10(-2) M) was observed only when the enzyme was inactivated with 10(-5) M p-CMB under 15 min. preincubation. Preincubation of the enzyme with 10(-4) M p-CMB for 45 min. did not result in its reactivation. Gel filtration of glutamine synthetase treated with 10(-4) M p-CMB has revealed the dissociation of the enzyme into inactive monomers. Incubation of glutamine synthetase with p-CMB at various pH values, incubation after pre-treatment with urea and experiments with HgCl2 indicate the presence of free and masked inside the globula SH-groups in the enzyme molecule. Competitive character of the enzyme inhibition with p-CMB with respect to ATP indicates that SH-groups of the active site participate in the ATP binding, probably, as Mg-ATP or Mn-ATP complexes. Data on the estimation of ionization constant of glutamate-binding group and experiments on the effect of histidine photooxidation on the enzyme activity indicate the presence of histidine residue in the enzyme active site, which participates in glutamate binding.     

Immobilized active monomers of D-glyceraldehyde-3-phosphate dehydrogenase from rabbit skeletal muscles and their coenzyme-binding properties

Experimental conditions favouring the dissociation of tetrameric rabbit muscle D-glyceraldehyde-3-phosphate dehydrogenase into active monomers were elaborated. The urea-induced dissociation of the tetramer was shown to be a stepwise process (in 2 M urea only dimers are formed; an increase in urea concentration up to 3 M causes the splitting of the dimers into monomers). The specific activity of immobilized monomers in the glyceraldehyde-3-phosphate oxidation reaction does not differ from that of the parent immobilized tetrameric form. The tetrameric enzyme molecule binds the coenzyme with a negative cooperativity (the first two NAD+ molecules bind with KD below 0.1 microM; for the third and fourth molecules the dissociation constant was determined to be equal to 5.5 +/- 1.5 microM (50 mM medinal buffer, 10 mM sodium phosphate, pH 8.2). The cooperativity of NAD+ binding is preserved in the immobilized preparation of tetrameric dehydrogenase. The immobilized monomers bind NAD+ with KD of 1.6 +/- 1.0 microM. The experimental results are consistent with the hypothesis according to which the association of catalytically active subunits into a tetramer changes their coenzyme-binding properties in such a way that the first two NAD+ molecules bind more firmly to a tetramer than to a monomer, whereas the third and the fourth NAD+ molecules bind less firmly.     

Cross-linked SecA dimers are not functional in protein translocation

The ATPase SecA is involved in post-translational protein translocation through the SecY channel across the bacterial inner membrane. SecA is a dimer that can dissociate into monomers with translocation activity. Here, we have addressed whether dissociation of the SecA dimer is required for translocation. We show that a dimer in which the two subunits are cross-linked by disulfide bridges is inactive in protein translocation, translocation ATPase, and binding to a lipid bilayer. In contrast, upon reduction of the disulfide bridges, the resulting monomers regain these activities. These data support the notion that dissociation of SecA dimers into monomers occurs during protein translocation.